Cross-linking reagents are well known and a considerable range of such reagents is available commercially. In broad terms, a cross-linking reagent comprises two or more reactive functional groups covalently linked together. The covalent linkage may be direct, but in many cases the reactive functional groups are spaced apart by respective covalent attachment to a spacer linkage. The reactive functional groups may be the same or may be different, (a heterofunctional cross-linking agent).
One application of cross-linking reagents is in the production of conjugate compounds which comprise a polypeptide or protein covalently attached to another chemical entity. Examples of such conjugate compounds include: antibody covalently attached to a signal producing chemical entity such as an enzyme or a chelated radioopaque or radioactive metal atom; antibody covalently attached to a cytotoxic chemical entity such as a toxin or a chelated radioactive metal atom; and an enzyme covalently attached to an antigenic analyte.
There is considerable interest in the use of cross-linking reagents for linking antibody or antibody fragments to signal-producing chemical entities or to cytotoxic chemical entities for use in diagnosis and cell-targeted therapy. It is known to produce such conjugate molecules using heterofunctional reagents which have a thiol-reactive functional group covalently linked to a different functional group such as an amino-reactive activated ester. Thus the heterofunctional cross-linking reagent is first reacted with the chemical entity to which it is desired to attach antibody. This may be done, for example, by reacting amino groups on the chemical entity with the amino reactive functional group on the heterofunctional cross-linking reagent. Next, free sulphydryl groups on the antibody or antibody fragment are reacted with the thiol-reactive functional group on the heterofunctional cross-linking reagent to yield the desired conjugate molecule.
Heterobifunctional cross-linking reagents (that is, heterofunctional cross-linking reagents with two reactive functional groups) are known which employ maleimides as the thiol-sepcific functional groups. An example of such a reagent is n-maleimido-benzoyl-N-hydroxysuccinimide ester (MBS) which has been used to prepare conjugate molecules comprising antibody or antibody fragments and enzymes or cytotoxic compounds. (O'Sullivan, M. J. et al, (1979), Anal. Biochem., 100, 100-108; Freytag, J. W. et al, (1984), Clin. Chem., 30, 417-420; Youle, R. J. et al, (1984), PNAS USA, 77, 5483-5486). Various derivatives of maleimide-based heterobifunctional cross-linking reagents are also known. The maleimide reagents are very reactive and readily hydrolyse to maleamic acid. They are, in general, of low specificity for sulphydryl groups, particularly when used at the pH and in the stoichiometric amounts that are often necessary for the preparation of conjugate molecules, to ensure that the resulting molecule has only one of each conjugated group.
Heterobifunctional cross-linking reagents are also known which employ .alpha.-halo acid derivatives as the thiol specific functional group. Whilst less reactive than the maleimide reagents, they are readily hydrolysed to the corresponding .alpha.-hydroxy acid derivatives and their tautomers. The aldehyde tautomers are essentially non-specific and reduce the overall specificity of the .alpha.-halo acid derivatives for thiol groups.
The low specificity of the known heterobifunctional cross-linking reagents can result in the production of complex mixtures of reaction products. The complex mixtures arise from non-specific reaction of the thiol reacting group with non-thiol groups of the antibody or the linked chemical entity. For example, the known thiol reacting groups commonly exhibit competitive reactivity for amine groups which can lead to complex polymerisation products. Unwanted side reactions are particularly disadvantageous where the linked chemical entity possesses secondary amines such as saturated ring nitrogens (e.g. in macrocyclic chelating ligands). Not only are these mixtures difficult to separate, but where the conjugate compound is for in vivo use, absolute purity is of paramount importance.
In the unconnected field of protein identification using HPLC it has been noted that 4-vinyl pyridine can be used as a specific marker for cysteine residues (Fullmer, C. S. et al, (1984), Anal. Biochem. 142, 336-339).